Matthew J. Linman,† Hai Yu,‡ Xi Chen,‡ and Quan Cheng*,† Department of Chemistry, University of California, Riverside, California 92521, and Department of Chemistry, University of California, Davis, California 95616
ABSTRACT Monitoring multiple biological interactions in a multiplexed array format has numerous advantages. However, converting well-developed surface chemistry for spectroscopic measurements to array-based high-throughput screening is not a trivial process and often proves to be the bottleneck in method development. This paper reports the fabrication and characterization of a new carbohydrate microarray with synthetic sialosides for surface plasmon resonance imaging (SPRi) analysis of lectin-carbohydrate interactions. Contact printing of functional sialosides on neutravidin-coated surfaces was carried out and the properties of the resulting elements were characterized by fluorescence microscopy and atomic force microscopy (AFM). Sambucus nigra agglutinin (SNA) was deposited on four different carbohydrate functionalized surfaces and differential binding was analyzed to reveal affinity variation as a function of headgroup sialic acid structures and linking bonds. SPRi studies indicated that this immobilization method could result in high quality arrays with RSD < 5% from array element to array element, superior to the conventional covalent linkage used for protein cholera toxin (CT) in a comparison experiment, which yields nonuniform array elements with RSD > 15%. Multiplexed detection of SNA/biotinylated sialoside interactions on arrays up to 400 elements has been performed with good data correlation, demonstrating the effectiveness of the biotin-neutravidin-based biointerface to control probe orientation for reproducible and efficient protein binding to take place. Additionally, the regeneration of the array surface was demonstrated with a glycine stripping buffer, rendering this interface reusable. This in-depth study of array surface chemistry offers useful insight into experimental conditions that can be optimized for better performance, allowing many different protein-based biointeractions to be monitored in a similar manner. KEYWORDS: carbohydrate microarray • surface plasmon resonance imaging • biotinylated sialosides • biointerface • biosensor
INTRODUCTION
C
arbohydrates are key components in cell surface interactions with both proteins and lipids, responsible for recognition, adhesion, and cell-to-cell signaling (1). Since they are structurally complex, there has been considerable effort in synthesizing carbohydrate analogs and investigating their structure-function properties. Monitoring multiple carbohydrate interactions is challenging but highly desirable for gaining affinity information in a timely and cost-effective manner, particularly for pharmaceutical research. This has led to the construction of carbohydrate microarrays for multiplexed detection of protein-carbohydrate interactions as early as in 2002 (2-5). Since then there have been a number of reports on carbohydrate microarrays, and the subject has been the topic for several reviews in the past few years (6-11). Several commercial instruments are available for fabrication of microarrays with varied quality. The methods for * To whom correspondence should be addressed. Tel: (951) 827-2702. Email:
[email protected]. Fax: (951) 827-4713. Received for review April 29, 2009 and accepted June 30, 2009 †
University of California, Riverside. University of California, Davis. DOI: 10.1021/am900290g
‡
© 2009 American Chemical Society
www.acsami.org
Published on Web 07/14/2009
array fabrication can generally be classified as either contact or noncontact printing. The former utilizes pin-type arrayers that transfer a defined volume of sample by directly touching the surface of the substrate, whereas the latter dispenses the sample droplets onto the substrate without a direct contact to the surface. The most common noncontact printing methods are photolithography and inkjet printing. Photolithography is very useful to pattern a variety of materials with different surface chemistries (12). However, it is incompatible with photosensitive compounds, including many biomolecules that cannot be deposited onto photoresists or metals. Inkjet printing, while cheaper than their contact printing counterparts, suffers from multiple drawbacks such as unsuitability to print on glass slides (13) and droplet smearing (14). Contact printing has been widely used and many pin-type arrayers are available, which are usually simpler in design, less expensive, and faster than noncontact printers (15). Contact printing has been used to pattern biomaterials, including DNA (16, 17), colloidal crystal arrays (18), and monoclonal antibodies (19), where elements can be densely packed in spatially defined areas. Recently several new types of contacting printing have emerged including dip pen nanolithography (DPN) (20) and varied forms of printing based on a PDMS continuous flow microfVOL. 1 • NO. 8 • 1755–1762 • 2009
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ARTICLE
Fabrication and Characterization of a Sialoside-Based Carbohydrate Microarray Biointerface for Protein Binding Analysis with Surface Plasmon Resonance Imaging
ARTICLE
luidic device (21). In spite of good flow control and small sample volume, one major drawback of patterning using microfluidics is that elaborate patterns with features more complex than simple cross patterned strips, such as arrays in high density, are hard to achieve without additional fabrication steps (22). The most popular method of contact printing employed currently is still the use of solid pins. There are a number of advantages to this technology including easy deposition of viscous solutions (23), simple design that enables reproducible and efficient printing (24), and simple cleaning procedure for the pins (24). One unique variant to the solid pin design is the pin and ring spotter employed in the GMS 417 arrayer, which uses a large ring to hold the sample and the pass of a pin deposits a droplet (25). The coefficient of variation (CV) using the GMS 417 Arrayer has been reported to be
